Project 2 (Structural Studies): Program Director/Principal Investigator: Moore, John P / Wilson, Ian A Abstract The goal of Project 2 in this HIVRAD proposal is to make significant advances toward the development of a HIV-1 vaccine that utilizes trimeric gp140 antigens (Env trimers). It is anticipated that such a vaccine would induce a substantial neutralizing antibody response. The role of Project 2 is to structurally characterize a wide range of SOSIP and related trimers as complexes with broadly neutralizing antibodies (bnAbs) and other ligands (receptors, small molecules) using X-ray crystallography, electron microscopy and other biophysical methods. In a truly integrative biophysical approach, we will determine the three-dimensional structures of Env trimer constructs to provide insights into design improvements intended to increase their stability, antigenicity and immunogenicity. The immediate goal of Project 2 is to capitalize on our recent exciting breakthroughs in generating stable soluble Env trimers (BG505 SOSIP.664, subtype A) that are amenable to high-resolution structure determination. We will increase the resolution of our current structural models and also determine new Env trimer structures based on a variety of genotypes (including from subtypes B and C). The resulting information will help us, working closely with Project 1 and Core A, to design a new generation of Env immunogens. To accomplish these goals, the Project 2 team will utilize a highly integrative structural biology and biophysical chemistry approach involving electron microscopy, X-ray crystallography, and a range of other biophysical tools (HDXMS, SEC-MALS, ITC, DSC, SPR), to generate high-resolution details of Env trimers and trimer- bnAb complexes. This structural information will not only provide a comprehensive view of the trimers but also define the epitopes for a diverse range of bnAbs, in the context of the trimer. This is important because many recently defined bNAb epitopes are quaternary in nature, or are otherwise not adequately described by crystal structures derived from complexes with simpler Env substrates such as gp120, gp120 outer domain, V1/V2 scaffolds, individual glycans, or linear peptides. Furthermore, we will delineate the structure and salient features of the glycan shield as the glycan moieties and composition on our SOSIP.664 trimers has been shown by our glycobiology collaborators to resemble those found on native, virion-associated trimers. Overall, we will use state-of-the-art structural and biophysical methods and our world-class structural biology facilities to generate high-resolution structures (~3-6 or better) for a variety of designed and engineered Env trimers of different genotypes, alone and in complex with bnAbs that define the main sites of vulnerability of Env. In addition, we will delineate the conformational changes that take place in Env as it transits from the closed, ground-state form to the open, fusion-active forms associated with CD4 and co-receptor binding.